The Ubiquitin Pathway
Ubiquitin-mediated proteolysis is an important pathway of non-lysosomal protein degradation which controls the timed destruction of many cellular regulatory proteins including, p27, p53, p300, cyclins, E2F, STAT-1, c-Myc, c-Jun, EGF receptor, IκBα, NFκB and β-catenin (reviewed in Pagano, 1997, FASEB J. 11: 1067). Ubiquitin is an highly conserved 76-amino acid polypeptide that is abundantly present in all eukaryotic cells. The ubiquitin pathway leads to the covalent attachment of a poly-ubiquitin chain to target substrates which are then degraded by the multi-catalytic proteasome complex (see Pagano, supra, for a recent review). Many of the steps regulating protein ubiquitination are known. Initially the ubiquitin activating enzyme (E1), forms a high energy thioester with ubiquitin which is, in turn, transferred to a reactive cysteine residue of one of many ubiquitin conjugating enzymes (Ubcs or E2s). The final transfer of ubiquitin to an e-amino group of a reactive lysine residue in the target protein occurs in a reaction that may or may not require an ubiquitin ligase (E3) protein. The large number of ubiquitin ligases ensures a high level of substrate specificity.
The Ubiquitin Pathway and the Regulation of the G1 Phase by F Box Proteins
Genetic and biochemical studies in several organisms have shown that the G1 phase of the cell cycle is regulated by the ubiquitin pathway. Proteolysis of cyclins, Ckis and other G1 regulatory proteins is controlled in yeast by the ubiquitin conjugating enzyme Ubc3 (also called Cdc34) and by an E3 ubiquitin ligase formed by three subunits: Cdc53, Skp1 and one of many F box proteins (reviewed in Patton, et al., 1998, Trends in Genet. 14:6). The F box proteins (FBPs) are so called because they contain a motif, the F Box, that was first identified in Cyclin F, and that is necessary for FBP interaction with Skp1 (Bai, et al., 1996, Cell 86:263). Cdc53 (also called Cul A) and Skp1 appear to participate in the formation of at least three distinct E3s, each containing a different FBP. Because these ligases are similar protein modules composed of Skp1, Cul A, and an FBP, they have been named SCF. The three SCFs identified in S. cerevisiae are: SCFCdc4 (which recruits the Ckis Sic1 and Far1, the replication factor Cdc6, and the transcriptional activator Gcn4, as substrates through the F-Box protein Cdc4), SCFGrr1 (which recruits the G1 cyclins Cln1 and Cln2 as substrates through the F-Box protein GRR1), and SCFMet30 (which recruits the G1 cyclin Cln3 as a substrate throughout the F box protein MET30; see Pagano and Patton, supra, for recent reviews).
The interaction of SCF ligase with its substrates occurs via the FBP. FBPs are present in all eukaryotes (at least 54 in mammals; Cenciarelli, et al., 1999, Current Biol. 9: 1177; Winston, et al., 1999, Current Biol. 9: 1180). In addition to the F Box, many FBPs contain additional domains that facilitate both protein:protein interactions, e.g. WD-40 domains or leucine-rich repeats (LRRs), and protein:DNA interactions, e.g. tankyrase binding domains or HNH domains. Since the substrate specificity of SCF ligases is dictated by different FBPs that act as substrate targeting subunits, the large numbers of FBPs with varying combinations of protein or DNA interaction domains ensure highly specific substrate recognition.
FBP1, A Mammalian FBP Involved in Regulation of APC/C
Fbp1, the mammalian homolog of Xenopus β-TrCP1 (β-transducin repeat containing protein) (Spevak, et al., 1993, Mol. Cell. Biol. 8:4953), was identified using Skp1 as a bait in a two-hybrid screen (Cenciarelli, et al., supra). Fbp1 is an F box protein containing seven WD-40 domains (Margottin, et al., 1998, Mol. Cell 1:565), and is involved in the degradation of IκBα family members in response to NFκB activating stimuli (Gonen, et al., 1999, J. Biol. Chem. 274:14823; Hatakeyama, et al., 1999, Proc. Natl. Acad. Sci. USA 96:3859; Hattori, et al., 1999, J. Biol. Chem. 274:29641; Kroll, et al., 1999, J. Biol. Chem. 274:7941; Ohta, et al., 1999, Mol. Cell 3:535; Shirane, et al., 1999, J. Biol. Chem. 274:28169; Spencer, et al., 1999, Genes Dev. 13:284; Winston, et al., 1999, Genes Dev. 13:270; Wu and Ghosh, 1999, J. Biol. Chem. 274:29591; Yaron, et al., 1998, Nature 396:590). In addition, consistent with the finding that Xenopus and Drosophila Fbp1 orthologs act as negative regulators of the Wnt/β-catenin signaling pathway (Jiang and Struhl, 1998, Nature 391:493; Marikawa and Elinson, 1998, Mech. Dev. 77:75), several studies report that human Fbp1 controls β-catenin stability in vitro and in mammalian cultured cells (Hart, et al., 1999, Curr. Biol. 9:207; Hatakeyama, et al., supra; Kitagawa, et al., 1999, EMBO J. 18:2401; Latres, et al., 1999, Oncogene 18:849; Winston, et al., 1999, Genes Dev. 13:270).
Well-characterized substrates of mammalian Fbp1 have been found to share a common destruction motif, DSGxxS, and are recognized by Fbp1 only upon phosphorylation of the two serine residues present in this motif. There is, however, some recent evidence for additional mammalian substrates of Fbp1 lacking a completely conserved binding domain, such as ATF4 (Lassot, et al., 2001, Mol. Cell. Biol. 21:2192), Smad3 (Fukuchi, et al., 2001, Mol. Biol. Cell 12:1431), NFκB p105 (Orian, et al., 2000, EMBO J. 19:2580) and NFκB p100 (Fong and Sun, 2002, J. Biol. Chem. 277:22111). A conserved DSGxxS motif is present not only in Fbp1 substrates but also in certain regulators of Fbp1, such as the HIV protein Vpu, which targets Fbp1 to the non-physiological substrate, CD4, in virally infected cells. (Margottin, et al., supra). The DSGxxS destruction motif may also be found in peptide regulators of Fbp1 termed pseudosubstrates; however, pseudosubstrates escape the normal degradation fate of other FBP target proteins and instead modulate the activity of the FBP, and corresponding Cks, such as cellular localization and substrate targeting. For example, the Fbp1 pseudosubstrate hnRNP-U not only inhibits Fpb1 from targeting inappropriate substrates but also serves to localize Fbp1 to the nucleus (Davis, et al., 2002, Genes Dev. 16:439).
A further level of complexity is added by the presence of a Fbp1/β-TrCP1 (beta-transducin repeat containing protein 1) paralogous gene product, called β-TrCP2 (beta-transducin repeat containing protein 2) or Fbxw1B (78% identical, 86% similar to β-TrCP1; Kipreos and Pagano, 2000, Genome Biology 1:3002.1). Fbp1 and β-TrCP2 are ubiquitously expressed in adult human tissues (Cenciarelli, et al., supra; Koike, et al., 2000, Biochem. Biophys. Res. Commun. 269:103). In addition, β-TrCP2 has biochemical properties similar to Fbp1 in its ability to sustain the ubiquitinylation of both β-catenin and IKBα family members in vitro and to control their degradation in mammalian cultured cells (Fuchs, et al., 1999, Oncogene 18:2039; Suzuki, et al., 1999, Biochem. Biophys. Res. Commun. 256:127; Tan, et al., 1999, Mol. Cell 3:527). Despite these similarities, Fbp1 localizes to the nucleus and β-TrCP2 localizes mainly to the cytoplasm (Davis, et al., 2002, Genes Dev. 16:439). It is not clear whether these two FBPs have overlapping functions in vivo, or if each of them recognizes specific substrates.
Deregulation of the Ubiquitin Pathway in Cancer and Other Proliferative Disorders
Cancer develops when cells multiply too quickly. Cell proliferation is determined by the net balance of positive and negative signals. When positive signals overcome or when negative signals are absent, the cells multiply too quickly and cancer develops.
Ordinarily cells precisely control the amount of any given protein and eliminate the excess or any unwanted protein. To do so, the cell ubiquitinates the undesired protein to tag the protein for proteasome degradation. This mechanism goes awry in tumors, leading to the excessive accumulation of positive signals (oncogenic proteins), or resulting in the abnormal degradation of negative regulators (tumor suppressor proteins). Thus, without tumor suppressor proteins or in the presence of too much of an oncogenic protein, cells multiply without control, forming tumors (reviewed by Ciechanover, 1998, EMBO J. 17: 7151; Spataro, 1998, Br. J. Cancer 77: 448). For example, abnormal ubiquitin-mediated degradation of the p53 tumor suppressor (reviewed by Brown and Pagano, 1997, Biochim. Biophys. Acta 1332:1), the putative oncogene β-catenin (reviewed by Peifer, 1997, Science 275:1752) and the Cki p27 (reviewed in Ciechanover, supra; Spataro, supra; Lloyd, 1999, Am. J. Pathol. 154: 313) have been correlated with tumorigenesis, opening to the hypothesis that some genes encoding ubiquitinating enzymes may be mutated in tumors.
Initial evidence indicates that human F box proteins play a role in the ubiquitination of G1 regulatory proteins as do their homologues in yeast. Unchecked degradation of cell cycle regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligase plays a role in the altered degradation of cell cycle regulators. A well understood example is that of Mdm2, a ubiquitin ligase whose overexpression induces low levels of its substrate, the tumor suppressor p53.
Alternately, F box proteins have been shown to interact directly with DNA regulating proteins or DNA itself. F box proteins in yeast are known to regulate genomic stability and senescence, and recent data has shown that F box inhibition in mammalian cells can lead to the loss of DNA damage checkpoints.
DNA Damage and the Cell Cycle
The Anaphase Promoting Complex or Cyclosome (APC/C) is a ubiquitin ligase that plays a crucial role in the regulation of mitosis and the G1 phase of the cell cycle (Peters, 2006). In early mitosis, APC/C is activated through binding to Cdc20, and in late M, Cdc20 is replaced by Cdh1, the second activator of APC/C. During G1, APC/CCdh1 remains active to ensure that certain positive regulators of the cell cycle do not accumulate prematurely. Then, at the G1/S transition, APC/CCdh1 is inactivated by phosphorylation to allow stabilization of its substrates and promote progression into S phase. Cdk1-cyclin A and Cdk2-cyclin A mediate the phosphorylation of Cdh1, resulting in the dissociation of Cdh1 from the APC/C core (Lukas et al., 1999; Mitra et al., 2006; Sorensen et al., 2001). Other mechanisms inhibiting APC/CCdh1 activity include Emi1 binding and degradation of both Cdh1 and Ubc10 (a ubiquitin conjugating enzyme that works with APC/C). During G2, both Cdh1 and Ubc10 reaccumulate, but APC/CCdh1 remains inactive due to CDK-dependent phosphorylation of Cdh1 and the presence of Emi1. In early mitosis, Emi1 is eliminated via the SCFβTrcp ubiquitin ligase, but the bulk of APC/CCdh1 remains inactive due to high Cdk1 activity. Ultimately, Cdh1 activation in anaphase involves Cdk1 inactivation by APC/CCdc20 and Cdh1 dephosphorylation. In yeast, this dephosphorylation is carried out by the Cdc14 phosphatase, but the mechanism in mammals remains unclear (D'Amours and Amon, 2004; Sullivan and Morgan, 2007).
Upon DNA damage, proliferating cells activate a regulatory signaling network to either arrest the cell cycle and enable DNA repair or, if the DNA damage is too extensive to be repaired, induce apoptosis (Bartek and Lukas, 2007; Harper and Elledge, 2007; Kastan and Bartek, 2004). The DNA damage response involves a number of factors that ultimately coordinate the spatiotemporal assembly of protein complexes at the site of DNA damage to initiate and maintain the checkpoint. Depending on the type of genotoxic stress, different checkpoint pathways are activated. UV and stalled replication forks activate the ATR-Chk1 pathway, whereas double-strand breaks result in the activation of both the ATM-Chk2 and the ATR-Chk1 pathways. After ATR is recruited to the site of damage, it phosphorylates and activates the effector kinase Chk1, a process requiring the mediator protein Claspin. Important downstream targets of Chk1 include p53 and Cdc25A, a transcription factor and an activator of Cdk1, respectively. Chk1-mediated phosphorylation induces the stabilization of p53 (with the consequent expression of the CDK inhibitor p21) and is required for the SCFβTrcp-mediated degradation of Cdc25A; thus, Chk1 activation results in the attenuation of Cdk1 activity, with the consequent inhibition of mitosis.
During the recovery from DNA replication and DNA damage stresses, the G2 checkpoint must be silenced. This process involves the degradation of Claspin via the SCFβTrcp ubiquitin ligase following the phosphorylation of Claspin by Plk1 (Mailand et al., 2006; Mamely et al., 2006; Peschiaroli et al., 2006). However, if DNA damage occurs during G2, SCFβTrcp-dependent ubiquitylation of Claspin is inhibited to re-establish the checkpoint. The blocking of Claspin ubiquitylation is at least partially due to the inhibition of Plk1, which occurs in response to DNA damage (Smits et al., 2000). In fact, Claspin is not phosphorylated on its degron and does not bind βTrcp in G2 cells that have been subjected to DNA damage (Peschiaroli et al., 2006). However, despite the lack of SCFβTrcp-Plk1-dependent ubiquitylation, Claspin continues to be ubiquitylated, only remaining stable due to a deubiquitylating enzyme (DUB), namely Usp28 (Zhang et al., 2006).
There is a general need for treatments relating to regulating or affecting the cell cycle to sensitize target cells, such as tumor or cancer cells, to cell death or apoptosis. In particular, there is a need for small molecule inhibitors that are useful as therapeutic agents, as well as for diagnostic and screening tools for identifying test therapeutic candidates.